Hawley R G, Covarrubias L, Hawley T, Mintz B
Proc Natl Acad Sci U S A. 1987 Apr;84(8):2406-10. doi: 10.1073/pnas.84.8.2406.
Retroviral vectors, designated handicapped, are described. These are genetically defective viruses that allow transfer of nonselectable genes under the transcriptional control of internal promoters. The basic handicapped vector (pHHAM) is derived from Harvey, Abelson, and Moloney murine retroviruses. It contains a 327-base-pair deletion in the 3' long terminal repeat that spans enhancer and promoter sequences. The deletion is successfully transferred to the 5' long terminal repeat after reverse transcription of viral RNA, yielding a provirus incapable of synthesizing viral transcripts. HHAM viruses containing the mouse c-myc gene under the control of immunoglobulin kappa chain gene regulatory elements, along with a selectable gene (neo) driven by a weak promoter (tk), were stably transmitted to cultured mouse B cells. The donor c-myc gene was transcribed from the kappa promoter in these cells. Helper-free virus-producing cell lines were generated at titers favorable for the efficient introduction of HHAM viruses, even without selection, into hematopoietic stem cells from mouse bone marrow. When returned to unirradiated congenic recipient mice, the cells were capable of long-term reconstitution of the myeloid and lymphoid lineages of W/Wv mutants and the lymphoid system of scid mutants.
描述了一种被称为缺陷型的逆转录病毒载体。这些是基因缺陷型病毒，可在内部启动子的转录控制下转移非选择基因。基本的缺陷型载体（pHHAM）源自哈维、阿贝尔森和莫洛尼小鼠逆转录病毒。它在3'长末端重复序列中包含一个327个碱基对的缺失，该缺失跨越增强子和启动子序列。病毒RNA逆转录后，该缺失成功转移至5'长末端重复序列，产生一种无法合成病毒转录本的前病毒。含有在免疫球蛋白κ链基因调控元件控制下的小鼠c-myc基因以及由弱启动子（tk）驱动的选择基因（neo）的HHAM病毒，被稳定地传递至培养的小鼠B细胞。供体c-myc基因在这些细胞中由κ启动子转录。即使不进行选择，也能产生无辅助病毒的细胞系，其滴度有利于将HHAM病毒高效导入小鼠骨髓的造血干细胞。当将这些细胞回输到未受辐射的同基因受体小鼠体内时，它们能够长期重建W/Wv突变体的髓系和淋巴系以及scid突变体的淋巴系统。
